Introduction.
We hope that this information will be useful to investigators who are
unfamiliar
with mouse breeding or who are breeding transgenic or gene targeted
mice
for the first time. These suggestions are based on our experience. They
are open to modification and should not be construed as a comprehensive
set of rules.

2. Mate mice when they are sexually mature (6 to 8 weeks old). We
recommend
that transgenic founders or chimeras be mated to C57BL/6 mice. After 6
generations of mating to C57BL/6, more than 99% of the genetic
background
will be C57BL/6. By analyzing gene expression on a C57BL/6 background,
any influence of the genetic background on gene expression will be
controlled
for by comparison to normal C57BL/6 mice. Alternatively, chimeras can
be
mated with 129/Sv+Tyr-c+p mice, which have the same genotype as the
embryonic
stem cells. This will produce mice with the targeted gene mutation on
the
129/Sv+Tyr-c+p background for comparison with the mutation on the
C57BL/6
background.

3. Expect litters within a month of mating since female mice go into
estrus every 3 or 4 days and the gestation time of mice is 19-21 days.
If no litters are produced after one month you should replace the mice
that you are mating with your founder. It is possible that the
transgenic
founder may be infertile due to consequences of transgene expression or
unknown reasons. It is possible that phenotypically male chimeras may
be
infertile because they are the result of colonization of a female
embryo
by male embryonic stem cells.

4. Reasons to use C57BL/6 mice.
a) C57BL/6 is a standard inbred strain, commonly used in
transgenic
breeding
b) mate 6 - 8 week-old mice for best reproductive performance
i. replace males when they are 1 year old
ii. replace females after 6 litters or when they are 6 months
old
c) mate a founder male with 2 females to get 2 litters in close
succession
d) mate a founder female with 1 male
e) mice usually mate again on the day the female gives birth,
resulting in a second litter 3 weeks
after the first.
f) to rapidly produce animals, rotate 2 females through a male's
cage every 1- 2 weeks
g) house pregnant females 1 or 2 per cage to prevent crowded
cages

5. Common problems and solutions:
a) female may not care for first litter
add proven breeder female to cage as helper and try again
b) female doesn't care for any litter
most often seen with 129 females kept on bedding which
precludes the construction
elaborate "subterranean" nests
129 mating cages should include nesting material at all
times
b) male may cannibalize litter
remove male from mating cage before female gives birth
c) fighting mice
i. separate the fighting mice, house them 1 per cage if
necessary
ii. females do not typically fight
iii. males may fight in the following circumstances:
male is placed in a cage containing other male(s)
male is separated at weaning and then reunited with male
littermates
male is weaned into a cage that contains males from
another
litter
males are aggressive and may begin to fight for no
apparent
reason
adult male attacks immature female when female is placed
in male's cage

6. Schedule for ear tagging, tail biopsies, weaning, and mating:
a) record births on the cage card and the pedigree
b) ear tag the pups when they are two weeks old
c) obtain tail biopsies as you apply ear tags
d) isolate tail DNA and determine genotypes before pups are 21
days old
e) record genotypes in the pedigree
f) wean pups when they are 21 days old
i. remove the pups from their mothers
ii. discard un-needed non-transgenic pups
iii. house males and females separately
f) when mice are 6 weeks old they may be mated (see 1. above)

Based on our experience, we propose the following guidelines for
chimera
breeding. If you have 10 or so chimeras to take through breeding then
these
guidelines apply. If you only have one or two animals then breed them
indefinitely
until you can produce additional animals from independent ES cell
clones.
You may be lucky get germline transmission at low efficiency (defined
as
1 agouti pup in 200 pups born). We recommend that you breed your
chimeras
to C57BL/6 partners. Pups produced from sperm derived from ES cells
will
have agouti coats, half of these animals should carry the targeted
gene.
Pups that are produced from sperm derived from the C57BL/6 host embryo
will have black coats.

1. Discard female chimeras unless you have high contribution female
chimeras and a distorted sex ratio favoring females (many female
chimeras
and few male chimeras). You probably injected a X:O cell line. The
females
may very will give germline transmission.

2. Breed as many males as you can, we have observed germline
transmission
from males with small quantities of agouti fur.

3. Breed males aggressively from the beginning ? rotate 2 females
through
the male's cage every two weeks. This will require 3 cages of 2 females
in addition to the male's breeding cage. Don't put the male chimera
into
a cage occupied by females, they may attack him.

5. If the chimeras don't produce pups by after 8 weeks of breeding
and
rotating females, they are infertile and should be discarded. If males
produce 6 or more litters without transmitting then they are not likely
to go germline and should be discarded.

Record Keeping:

1. Establish a Pedigree for each founder animal or offspring that is
mated.

2. Have customized cage cards printed for mating cages and weaning
cages.
Mating cage cards should provide an instant summary of the activity in
the cage, and serve as permanent records. The weaning card allows rapid
location of mice according to line and ear tag.

3. Use a customized log sheet to record ear tag number, sex, coat
color,
tail DNA log number, and genotype for each mouse that is tagged and
tailed.
Keep the log sheets with the pedigree.

Periodically we receive requests to help rescue mouse strains that are
no longer breeding. Often the mice are beyond their preak reproductive
years. Here are some of the approaches that can be used to "rescue" old
strains.

1. Use C57BL/6 F1 mice to set up new breeding cages (see mouse breeding
suggestions above). Most C57BL/6 F1 hybrids have good
reproductive performance. We prefer (C57BL/6J X DBA/2J)F1
animals. Outbred CD-1 and ICR mice can be used, but introduce more
genetic complextiy to the line.

2. Follow the breeding females for copulation plugs. Females enter
estrus every 3 days or so. Mate the animals on Monday and follow them
for plugs. If they haven't plugged by Friday, keep following them over
the weekend until they plug or unmate then and re-mate on Monday.
Alternatively, the Transgenic Core can mate superovulated F1 female
mice to your male(s) in a rederivation
procedure. Eggs are collected from the females and fertilization is
determined under the microscope. Fertilized eggs are transferred to
pseudopregnant females to obtain pups. If the male doesn't generate
fertilized eggs (no plugs or plugs without fertilization then the next
step is in vitro fertilization (see #3).

3. If the F1 females plug but don't become pregnant then the males you
are using to rescue the line are subfertile, by definition. We can use
euthanize the male(s), collect sperm and perform an
in vitro fertilization (IVF) procedure. The Transgenic Core
routinely does IVF with excellent results. Once we rescued a line from
the last male even though it died two days before the IVF. In this
case, the dead male was stored in a sealed plastic bag (to prevent
dessication) in the refrigerator until the IVF. Another time we rescued
a mouse strain from a single spontaneous male mutant with ectopic
caudal limbs in the place of a penis (polypodia mutation - Ppd
gene on the X chromosome).

4. If the F1 males don't plug the females then females are most likely
anovulatory. In this case you can send the old females to the Jackson
Laboratory for ovarian transplantation into a immunocompromised female
(to prevent graft rejection) and you may be able to recover the line
this way. The Transgenic Core does not offer this service.

5. If you took the precaution of cryopreserving
the line as frozen eggs, the Core can thaw out the eggs and recover
the line. If sperm were cryopreserved we may or may not be able to
recover the line by IVF or ICSI, depending on the genetic background of
the sperm. We recommend that frozen sperm be tested for successful IVF
before a line is discontinued.

6. If the line was derived from an ES cell clone, it can be re-injected
to generate a new set of germline chimeras.

7. If the line is available from another laboratory you can import
them.

Three transgene (Tg) transmission patterns occur in transgenic
founder
animals. Most founder animals transmit their Tg to 50% of their
offspring.
About 10% to 20% of founders are mosaic for the Tg due to late Tg
integration
during embryogenesis. A variable proportion of founders (5% to 30%)
have
more than one Tg integration site. Transmission to 30% or less of
offspring
is a sign that the founder's germ cells are mosaic for the Tg. In these
cases it is advisable to verify the transgenic status of the founder
and
to breed the mouse as efficiently as possible. Founders with more than
one Tg integration may transmit the Tg to 80% or more of their
offspring.
Different insertion sites usually segregate independently. Southern
blot
analysis of these offspring is used to group mice have according to
insertion
site. This simplifies analysis of transgene expression since expression
patterns associated each independent insertion are isolated from each
other.
Offspring of transgenic founders transmit the Tg as normal Mendelian
gene,
regardless of whether the founder was mosaic or multi-integrant.

Gene Targeting Transmission Patterns:

Two transmission patterns occur in embryonic stem (ES) cell-mouse
chimeras.
Either they transmit or they don't. The transmission ratios we have
observed
vary from 100% to 0.5%. Infertile male chimeras may also result. Since
the ES cells are XY only male chimeras should be bred. We recommend
that
you take a male chimera through six litters before deciding to give up
on it. If a chimera has a high transmission ratio, you should consider
mating it directly with females from the 129 mouse strain that matches
the origin of the ES cell line to obtain your targeted gene mutation on
an inbred background.

Hemizygotes or Homozygotes?

We recommend that transgenic lines be maintained as hemizygotes. The
primary disadvantage of maintaining lines as hemizygotes is that all
offspring
need to be genotyped to determine which are transgenic. Establishment
of
homozygous transgenic lines is is costly and entails additional
breeding
to produce the first homozygotes. Additional test breeding is needed to
ensure homozygosity. Additional time is required to establish a
homozygous
line from a mosaic founder with limited transgene transmission. Extra
breeding
will be necessary to establish homozygous lines for each integration
site
in founders with multiple integrations. If this is not done carefully,
animals with combinations of different integration sites will result.
This
will complicate interpretation of experimental results since different
integration sites can have different transgene expression patterns.
About 5% of transgene integration events interrupt an endogenous gene
important
for normal development (Meisler
MH. 1992. Trends Genet. 8:341). Affected mice may display a
phenotype unassociated
with the transgene which may obscure the effects of transgene
expression.
Unequal crossing-over between transgene arrays in homozygotes may
result
in transgene rearrangement which will affect expression. This kind of
instability
occurs less often in hemizygotes. Many other difficulties can be
avoided
by maintaining hemizygous transgenic lines.

Table 1 is adapted from Manipulating the Mouse Embryo: A Laboratory
Manual, by B. Hogan, R. Beddington, F. Constantini, and E. Lacey,
published
by Cold Spring Harbor Laboratory. This book has an excellent
bibliography
for information about mouse strains and breeding.

An excellent source is the second edition of Biology of the
Laboratory
Mouse, by the Staff of the Jackson Laboratory, published by
Blakiston
Division of McGraw-Hill.

Some investigators will need to know the alleles at various
polymorphic
loci carried by the transgenic founders. This information can be found
by looking up the genotypes of the mice used to obtain fertilized eggs
for microinjection in:

Genetic Variants and Strains of the Laboratory Mouse, eds. M.F. Lyon
and A.G. Searle, Chapter 17; "Strain Distribution of Polymorphic
Variants"
by T.H. Roderick and J.H. Guidi, published by the Oxford University
Press.